Construction Of Fluorescence Labeled GPR119 Cell Line

GPR119 belongs to the family A of G protein-coupled receptors and most expresses in intestinal endocrine cells and the surface of pancreatic β cell L. The L cells can directly promote the secretion of GLP-1 when GPR119 is activated, and induced all the physiological functions of GLP-1, but also directly promote β cells secrete insulin. Therefore, GPR119 research becomes a new target for the treatment of diabetes in recent years. Based on GPR119 molecular pharmacology and biological characteristics, the establishment of appropriate drug screening model for small molecule of GPR119 agnoist and finding the new way of regulating anti-diabetic agent is developed. This study will focus on establishment of fluoresce directly or indirectly labeled GPR119 cell lines for screening dugs from natural products (especially micromolecule compounds).This study first constructed the eukaryotic expression vector of GPR119 with green fluorescent protein (GFP) and transfected it into human osteosarcoma U2OS cells and HEK293 and obtained fluorescently labeled transfected cells of GPR119. However, during resistance screening process all transfected cells were dead, no stable recombination cells has been esctablished until now. Various solutions, including the replacement of cell types, adding calcium inhibitor, lower concentration of plasmid were used to dissolve this problem, but the result is same as before.Maybe the large molecular weight of GFP directly labeled to GPR119 affect its structure, resulting in difficulties in establishment of fluorescently labeled GRP119 cell lines. The β-arrestin is an important adapter protein and regulation of signal transduction, as an important role in the signal transduction of G protein-coupled receptors (G-protein-coupled receptor, GPCR), and it specifically activated by ligand binding to the receptor, and therefore the polymerization can be detected by a fluorescence-labeled β-arrestin with GPR119. So, using the constructed β-arrestin fluorescently labeled cells, native GPR119 expressed cell line has been established for drug screening. In our experiments, GPR119 was inserted into plasmid pCMV6-A-Hygro to construct pCMV6-A-Hygro-A-GPR119, then ransfected into fluorescently labeled β-arrestin cell lines. When agonist was added into media, fluorescence aggregation was observed.In this study, GPR119 was direct and indirect labled by GFP. When GRP119 was directly labled, the self-aggregation of fluorescent was observed in transfected cells and lead to fail in form cell lines. In indirectly labled with β-arrestion the transfected cell can be active by agonist and aggregated. Our research provided a new method to explore screening new GRP119 agonist from natural products.